Seeinstructions): Yersinia pestis and Francisella tularensis are the causative agents of plague and tularemia, respectively. Both of these bacteria are extremely virulent for humans when aerosolized and thus have the potential for use as agents of bioterrorism. Greater knowledge of the molecular mechanisms underlying Y. pestis and F. tularensis pathogenesis is urgently needed. The goals of this proposal are to identify Y. pestis andF. tularensis virulence factors and elucidate mechanisms of virulence factor biogenesis and function. Y. pestis contains ten chaperone/usher (CD) secretion pathways, only two of which have been characterized. CU pathways are utilized by a broad range of bacteria for the assembly of virulence-associated surface structures such as the hair-like fibers termed pili. Y. pestis expresses pilus fibers on its cell surface. The first aim of this proposal will characterize the functions of the Y. pestis CU pathways in expression of the pili and determine their roles in virulence. The molecular basis for the high infectivity and virulence of F. tularensis is just beginning to be understood. The pil genes present in F. tularensis are required for the assembly of pili and for protein secretion into the extracellular medium. These genes are virulence factors of F. tularensis. The second aim of this proposal will determine the mechanism of the F. tularensis pil genes in both pilus assembly and protein secretion, and will define their functions in pathogenesis. The TolC protein is also a virulence factor of F. tularensis. TolC forms an outer membrane channel that participates in secretion systems for the export harmful molecules and proteins such as toxins. The third aim of this proposal will investigate the mechanism of TolC-related secretion systems in the pathogenesis of F. tularensis, and will determine if TolC is required for the secretion of a F. tularensis toxin that modulates the host response to infection. This proposal will be done as part of a Program Project Grant, entitled "Agents of Bioterrorism: Pathogenesis and Host Defense." The Yersinia experiments will be performed in collaboration with Project 1 (Bliska) and the Francisella experiments will be peformed in collaboration with Project 2 (Furie). This Program Project Grant will produce a synergistic effort that will broaden our understanding of bioterrorism agents and advance fundamental knowledge of microbial pathogenesis. RELEVANCE (See instructions): The work described in this proposal will identify and characterize virulence factors of the potential Dioterrorism agents Yersinia pestis and Francisella tularensis. This information will elucidate mechanisms of pathogenesis and create opportunities for the development of novel methods to detect, prevent and treat outbreaks of plague or tularemia.